This study investigated the feasibility of combining radionuclides with photothermal agents in the diagnosis and treatment of ATC.Anaplastic thyroid cancer (ATC) cells cannot retain the radionuclide iodine 131  by the standard chloramine T method. 131I-HSA and indocyanine green (ICG) were non-covalently bound by a simple stirring to obtain 131I-HSA-ICG nanoparticles. Characterizations were performed in vitro. The cytotoxicity and imaging ability were investigated by cell/in vivo experiments. The radio-photothermal therapy efficacy of the nanoparticles was evaluated at the cellular and in vivo levels.In vivo fluorescence imaging and single-photon emission CT (SPECT)/CT imaging in small animals confirmed that I-HSA-ICG/131I-HSA-ICG nanoparticles could stay in tumor tissues for 4–6 days. Compared with other control groups, 131I-HSA-ICG nanoparticles had the most significant ablation effect on tumor cells under the irradiation of an 808-nm laser.The synthesized nanoparticles had a suitable size (25–45 nm) and objective biosafety. Under the irradiation of near-IR light, the photothermal conversion efficiency of the nanoparticles could reach 24.25%. 131I-HSA-ICG nanoparticles could successfully perform dual-modality imaging and treatment of ATC, which provides a new direction for the future treatment of iodine-refractory thyroid cancer.In summary,  Studies had shown that a slight temperature increase could increase the vascular permeability of the tumor, initiate the recruitment of certain biomolecules or cells, and promote the spread of photothermal agents in the tumor . TemperaHuman serum albumin (HSA) molecules were widely used as nanocarriers due to the following advantages: excellent blood retention activity, non-immunogenicity, biocompatibility, high water solubility, biodegradability, chemical stability, and glomerular filtration , 11. HSA131I to construct an easily retained, safe, and harmless multifunctional NP for locally combined photothermal and radionuclide therapy on iodine-refractory thyroid cancer. The main scheme of our study is shown in Therefore, the goal of this study is to combine ICG with 131I was bought from Tianjin Atom Technology Co., Ltd. . Chloramine T was purchased from C&S Biochemical Technology Co., Ltd. . Sodium sulfite was purchased from the Beijing Procurement and Supply Station of China Pharmaceutical Company, and sodium iodide (NaI•2H2O) was purchased from Bide Pharmaceutical Technology Co., Ltd. . Methyl thiazolyl tetrazolium (MTT) was purchased from SDN Company. Dimethyl sulfoxide was purchased from Solebold Technology Co., Ltd. . Fetal bovine serum (FBS) was purchased from M&C Co., Ltd. Gibco Dulbecco’s modified eagle’s medium, 1640 medium, and pancreas Protease digestion solution [ethylene diamine tetraacetic acid (EDTA)] were purchased from Hai Klonn Biochemical Co., Ltd. (USA). Calcein acetoxymethyl ester  and propidium iodide (PI) were purchased from Dongren Chemical Technology Co., Ltd. . Physiological saline was purchased from Chinese Otsuka Pharmaceutical Co., Ltd. Five percent chloral hydrate was purchased from the General Hospital of Tianjin Medical University . Ultrapure water was purchased from Wahaha Group Co., Ltd. . Ki67 antibody was purchased from FabGennix . The urine iodine quantitative detection kit (AR 60T) was purchased from Wuhan Zhongsheng Biochemical Technology Co., Ltd. . TLC Scanner was purchased from Hengyide Technology Co., Ltd .All of the reagents used in the research were of analytical grade. ICG was bought from Aladdin Biochemical Technology Co., Ltd. . HSA was offered by Dingguo Biotechnology Co., Ltd. . Na2O was dissolved. The HSA, chloramine T, and NaI•2H2O solutions were mixed for 1 min. Then 1 ml of sodium metabisulfite solution  was added to stop the reaction. The reaction solution was ultrafiltered by a centrifuge  3 times . Next, according to the molar ratio of HSA : ICG = 1:1, the corresponding concentration of ICG was calculated and added to the mixture. At the same time, another portion of HSA was taken separately and reacted with ICG. The final mixtures were stirred by a magnetic stirrer  for 1 h and ultrafiltered 3 times . Finally, the synthesized HSA-ICG and I-HSA-ICG NPs were lyophilized and stored away from light. In addition, the NaI•2H2O could be replaced with a certain unit of Na131I to synthesize 131I-HSA-ICG NPs with the same method.To avoid radioactive damage and contamination, we synthesized non-radioactive I-HSA-ICG NPs for characterization experiments. First, HSA was labeled with iodine by the standard chloramine T method. In 1 ml of phosphate-buffered saline (PBS) (0.01 mol/ml), 20 mg of HSA, 5 mg of chloramine T, and 5 mg of sodium metabisulfite were dissolved separately, and in 1 ml of water, 930 mg of NaI•2H−1) of I-HSA-ICG NPs were recorded by FT-IR spectrometer . The UV–visible–NIR absorption spectra of I-HSA-ICG NPs were measured by UV-3600 plus spectrophotometer . In addition, static stability observations were performed on ICG and I-HSA-ICG. Both NPs were dissolved in ultrapure water, PB , FBS, and Gibco 1640 medium. The solutions were allowed to stand for 14 days at room temperature. Photos were taken at different time points . During the 14-day observation period, the particle sizes of I-HSA-ICG NPs and the ICG release from I-HSA-ICG NPs in H2O and PBS were monitored. In addition, dynamic radioactivity monitoring of 131I-HSA-ICG NPs was subjected for 8 days to thin-layer chromatography to evaluate the release of free 131I.The hydration diameter and zeta potential of the I-HSA-ICG NPs were measured by a Malvern NP size analyzer . The newly obtained I-HSA-ICG NP suspension was transferred to a 200-mesh copper mesh coated with carbon and stained with 2% (weight/volume) phosphotungstic acid. It was dried at room temperature and analyzed by transmission electron microscope (TEM)  to obtain the particle size. With pure potassium bromide as the background, the Fourier transform-IR (FT-IR) spectra : ICG, HSA-ICG NPs, and I-HSA-ICG NPs. Group 2 included I-HSA-ICG NPs with different concentrations. The control group was water. The above solutions were irradiated with 808-nm lasers . During the irradiation, the IR calorific value camera  was used to record the calorific value images of each solution, and the temperatures were recorded every 30 s. After irradiation, the I-HSA-ICG solution was left to stand and centrifuged every 30 min. The supernatant was taken, and an equal volume of background solution was added to the stock solution. According to the principle of iodine-catalyzed arsenic–cerium reaction, the iodine release of I-HSA-ICG was monitored within 3.5 h after illumination through the instructions of the urine iodine quantitative detection kit.Two experimental groups were established. Group 1 included the following solutions with the same concentration of ICG (C2) was used to irradiate water (1 ml) and I-HSA-ICG solution . The irradiation was stopped after the temperature of the solution no longer increased. The initial ambient temperature was reached naturally. Temperatures were recorded every 30 s during heating and cooling. The light-to-heat conversion efficiency (η) of I-HSA-ICG NPs was calculated by the following formula .The human undifferentiated thyroid carcinoma cell lines ARO and FRO were kindly provided by Dr. Shunichi Yamashita and Dr. Norisato Mitsutake . ARO, FRO, and mouse breast cancer cells (4T1 cells) were plated in 96-well plates at a density of 1 × 10Group 1 formulated HSA-ICG NPs into gradient concentrations  and incubated them with FRO cells and 4T1 cells for 24 h. In group 2, HSA-ICG/I-HSA-ICG NPs were formulated into gradient concentrations  and incubated with ARO cells for 24 h. Finally, cell viability was obtained by the standard MTT method.3 cells/well. On the next day, the cells were treated with Na131I and 131I-HSA-ICG NPs in different radioactivity gradient counts  and co-cultured for 24 h. Cell survival rates were measured by the standard MTT method.ARO cells were seeded in a 96-well plate at a density of 3 × 10131I-HSA-ICG NPs were co-cultured with ARO cells in 96-well plates for 24 h. Each well of different groups was irradiated by an 808-nm laser with different densities for 5 min. The control solution was PBS. The radioactive dose of each well was 500 μCi. Cell survival rates of each group were acquired by the standard MTT method.Different concentrations of I-HSA-ICG/131I-HSA (500 μCi/well), and 131I-HSA-ICG (500 μCi/well) for 24 h. Cells in groups 4, 5, and 6 were respectively co-cultured with PBS, HSA-ICG, and 131I-HSA-ICG (500 μCi/well) for 24 h and then irradiated by an 808-nm laser (2.5 W/cm2) for 5 min. Among them, the content of ICG in each group was 11.7 ppm. The cell viability of each group was determined by the standard MTT method. A 1:1 ratio of PI and Calcein-AM mixture was prepared. The above procedures were repeated, and live/dead cell staining of the mixture was carried out after the end of the treatment. Fluorescence images of dead and live cells were acquired by an inverted fluorescence microscope.ARO cells were treated with different methods. Cells in groups 1, 2, and 3 were respectively co-cultured with PBS, 131I during treatment. The animal experiments were conducted in accordance with the Tianjin Medical University Institutional Animal Care and Use Committee guidelines.The animals used in this study were male BALB/c nude mice aged 4–5 weeks and weighing 15–18 g . ARO cells were planted under the skin of the right/left side of the mouse thigh. Mice were treated when subcutaneous tumors had grown to about 6 mm in diameter. One week before treatment, all nude mice in the radionuclide treatment group were given 1% NaI aqueous solution, and 1 ml of 1% NaI aqueous solution was intraperitoneally injected 1 day before treatment. The aim was to block the thyroid glands of nude mice and avoid uptake of radioactive Male Kunming mice  aged 4–5 weeks were randomly divided into 2 groups, with 5 mice in each group. Group 1 was injected with 100 μl of PBS through the tail vein; group 2 was injected with 100 μl of I-HSA-ICG solution (5 mg/ml) through the tail vein. During the 30-day observation period, the body weight changes of the mice at different time points were recorded. After 30 days, the heart, liver, spleen, lung, and kidney of the mice were removed for H&E staining. The eyeball blood samples of the mice were taken for biochemical examination.ICG = 100 ppm), respectively. Intravital fluorescence imaging was then performed. Groups 3 and 4 were injected intratumorally with 100 μl of Na131I and 131I-HSA-ICG NPs (800 μCi/mouse), respectively. Then single-photon emission CT (SPECT)/CT imaging  was performed. By delineating regions of interest on SPECT/CT slices, the radioactivity counts of tumor, liver, kidney, and lung were monitored at different time points within 8 days.The tumor-bearing nude mice were randomly divided into 4 groups with 3 mice in each group. Groups 1 and 2 were injected intratumorally with 100 μl of pure ICG and I-HSA-ICG NPs (C131I and 131I-HSA-ICG NPs (800 μCi/mouse), respectively. Groups 3, 4, and 5 were injected intratumorally with 100 μl of PBS, HSA-ICG NPs, and 131I-HSA-ICG NPs (800 μCi/mouse), respectively, followed by 808-nm laser irradiation for 10 min. The ICG content of all experimental groups was 40 ppm. During the irradiation period, a calorific value camera was used to record and photograph the local temperature increase of the irradiated part. All groups were observed for 17 days after the end of all treatments. Changes in body weight and tumor volume of nude mice at different time points were recorded and photographed. After 17 days, the tumors of the mice were removed and weighed. Six days after treatment, Ki67 antibody immunohistochemical staining was performed on tumor tissues of each group according to the instructions.Tumor-bearing nude mice were randomly divided into 5 groups with 3 mice in each group. Groups 1 and 2 were injected intratumorally with 100 μl of Nap-value was less than 0.05, it represented a significant statistical difference (p < 0.05).The data were all expressed as mean ± SD. The differences between groups were compared by independent-samples t-test or two-tailed t-test. When the In vitro characterizations were performed to confirm our successful synthesis of the NPs. I-HSA-ICG NPs were spherical with a diameter range of 25–45 nm observed by TEM (−1 (mainly caused by C═O stretching vibration). Amide II band appeared between 1,500 and 1,600 cm−1 (because CN stretching vibration was accompanied by NH bending mode). The amide III band appeared at 1,200–1,300 cm−1 (mainly caused by NH bending mode and CN tensile vibration) (−1) and amide II . Both of the peaks correspond to the characteristic peaks in free HSA. In addition, the zeta potentials of HSA-ICG and I-HSA-ICG NPs at different concentrations in PB buffer and water averaged about −3 and −16 mV (d by TEM . The prebration) –27. Fig2O and PBS was periodically monitored. The results showed minimal ICG shedding from I-HSA-ICG, even with slight fluctuations at the 72-h time point  to irradiate water and different concentrations of I-HSA-ICG solutions, it was found that an ideal photothermal effect could be achieved when the concentration was 4 mg/ml . As shown in 131I-HSA-ICG + light group was about 20%, indicating that the experimental group had a significant therapeutic effect at the cellular level. ARO cells of different treatment groups were carried out using Calcine-AM and PI solution to live/dead cell dye. Green represents living cells, and red represents dead cells. The results confirmed that the ARO cell mortality rate in the photothermal and radioactive treatment group was the highest   and a ciin vivo. As a low-toxic and safe clinical dye, ICG had a high affinity for proteins (131I therapy (RAI) was the mainstay of standard treatment for DTC  could effectively synergize radioactive iodine therapy and chemotherapy in animal tumor models. They found that changing PTX to MnO2 could also improve the efficacy . The in vitro and in vivo experiments confirmed that the NPs have objective biological safety and high stability. Compared with pure ICG and Na131I, 131I-HSA-ICG NPs could be retained in the tumor for a long time, which might be related to the enhanced permeability and retention of NPs  treatment combined with continuous 131I radiation could cause significant damage or even death to cancer cells. Compared with ICG, Na131I, and HSA-ICG NPs, the tumors in the 131I-HSA-ICG NPs treatment group were significantly damaged or even ablated after 808-nm laser irradiation. Therefore, the combination therapy efficacy of radionuclide 131I and ICG is significantly better than that of single therapy.Therefore, this study combined ICG and n of NPs . ICG hign of NPs , 54. A tn of NPs , which m minutes , 56. Cel131I-HSA-ICG NPs could stay in tumor cells for a long time, which constantly helped in using radioactive radiation to induce cell damage during and after PTT, thereby further enhancing the tumor ablation rate. This method provides an optimization strategy for the combined treatment of HSA, and this platform can be used to further develop other types of nanomaterials to optimize efficacy in the future.In conclusion, our work combined radionuclide therapy with PTT to construct multimodal imaging and therapy integrated nanomaterial for diagnosis and treatment. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Experimental Animal Ethics Committee of Tianjin Medical University.Conceptualization: ZM. Methodology: XZ and ZM. Software: QJ and YS. Validation: XZ and ZY. Formal analysis: XZ. Investigation: XZ and ZY. Resources: ZM, QJ, YS, and YJ. Data curation: XZ. Writing—original draft preparation: XZ and ZY. Writing—review and editing: ZM. Visualization: XZ. Supervision: ZM and NL. Project administration: ZM. Funding acquisition: ZM, YJ. All authors contributed to the article and approved the submitted version.This study was supported by the China National Natural Science Foundation grant 81571709 and 81971650, the Tianjin Science and Technology Committee Foundation grant 21JCYBJC01820, the Key Project of Tianjin Science and Technology Committee Foundation grant 16JCZDJC34300, and the Project of Tianjin Science and Education Commission grant 20JCQNJC01610. This study was supported by the Tianjin Medical University General Hospital New Century Excellent Talent Program, the Young and Middle-aged Innovative Talent Training Program from Tianjin Education Committee, and the Talent Fostering Program (the 131 Project) from Tianjin Education Committee, Tianjin Human Resources, and Social Security Bureau (awarded to ZM). This study was supported by Tianjin Medical University General Hospital (the Departments of Nuclear Medicine and Radiology).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Few tools exist for the prediction of new-onset AF (NOAF) after myocardial infarction (MI) and its relation to IS. We studied the usefulness of CHA2DS2-VASc in predicting NOAF and IS in a long-term follow-up after MI. Consecutive MI patients without baseline AF , discharged from 20 hospitals in Finland during 2005–2018, were retrospectively studied using national registries. The outcomes of interest after discharge were NOAF- and IS-assessed with competing risk analyses at one and ten years. The median follow-up was 4.2 years. The median baseline CHA2DS2-VASc score was 3 (IQR 2–5). The likelihood of both NOAF and NOAF-related IS increased stepwise with this score at one and ten years . The one-year-adjusted subdistribution hazard ratio (sHR) was 4.03 (CI 3.68–4.42) for NOAF in patients with CHA2DS2-VASc scores ≥6 points. The cumulative incidence of IS was 15.2% in patients with NOAF vs. 6.2% in patients without AF at 10 years after MI . Coronary artery bypass surgery was associated with a higher NOAF incidence compared to percutaneous coronary intervention . The CHA2DS2-VASc score is a simple tool used to estimate the long-term risk of NOAF and IS after MI in patients without baseline AF. Coronary bypass surgery is associated with an increased NOAF incidence after MI.The CHA Patients suffering from myocardial infarction (MI) are known to be at an increased risk of atrial fibrillation (AF) ,2. New-on = 20, including five university hospitals with coronary surgery available) were included [Data on all consecutive MI patients admitted to participating hospitals in Finland between 1 January 2005 and 30 June 2018 who were discharged alive were retrospectively collected from the Care Register for Health Care in Finland (CRHC). All Finnish hospitals that treat MI patients , as well as one additional point for those with congestive heart failure, hypertension, being aged 65–75, diabetes, and the female sex. Two additional points were calculated for those aged ≥75 and with stroke/TIA/ischemic thromboembolism. Revascularization procedures during index MI hospitalization and the type of MI (ST level changes) were recorded [Baseline congestive heart failure, hypertension, diabetes, and stroke/transient ischemic attack/ischemic thromboembolism were detected  Supplem. Age at recorded .The outcomes of interest were NOAF and NOAF-related IS assessed at a one- and ten-year follow-up after index event (MI for NOAF and NOAF for NOAF-related IS). The outcomes are detailed in the The CRHC registry data, including data on all hospital admissions and major interventional procedures, and special reimbursement entitlement data were obtained from Findata (permission no: THL/164/14.02.00/2021). Pharmacy purchases of oral anticoagulants before MI were obtained from a national drug purchase database (THL/164/14.02.00/2021). Mortality and cause-of-death data were obtained from Statistics Finland (TK-53-484-20). The included registries are mandated by law and cover the entire Finnish population. Ethical board review and informed consent were waived due to the study design, and the participants were not contacted. The legal basis for the processing of personal data is public interest and scientific research , Article 6(1)I and Article 9(2)(j); Data Protection Act, Sections 4 and 6).p-value < 0.05. Analyses were performed with SAS version 9.4 .The outcomes were studied using the cumulative incidence function and Fine–Gray regression, accounting for the competing risk of non-endpoint-specific death . Schoenf2DS2-VASc score was 3 . Percutaneous coronary intervention (PCI) was performed on 55.3% of the patients and a coronary artery bypass (CABG) was performed on 7.5%, while 37.3% were treated conservatively . An increased CHAp < 0.0001) and ten years . Conversely, CABG was associated with an increased NOAF incidence compared to non-revascularized patients at both the one-year  and ten-year  time points. Compared to patients revascularized with PCI, CABG was associated with a higher incidence of AF at one year  and at ten years  after MI. The incidence of NOAF within one year after MI did not change significantly during the study period  and multivariable  analyses of the follow-ups of all study patients. NOAF was diagnosed simultaneously with IS in 4.1% of all NOAF patients and in 46.1% of NOAF-associated IS cases, with a median time of six days (IQR 0 days–1.7 years) between NOAF diagnosis and IS. The cumulative incidence of IS was 6.4% at one year and 10.9% at ten years after NOAF diagnosis. A higher CHA2DS2-VASc score was associated with a higher proportion of NOAF-associated IS (trend p = 0.001)  at one year and 1.85 (1.31–2.62) at ten years compared to the lowest score group (p = 0.007), but there was no difference in the CABG and no-revascularization groups . Of all IS cases, 853 (22.5%) were associated with NOAF. In addition, NOAF was associated with an increased IS occurrence both in univariable  . The incre group . Revascu2DS2-VASc score is a feasible tool used to predict NOAF during a long-term follow-up in patients with a history of MI. The hazard of developing NOAF within one year after MI was 4.6-fold higher in those with the highest CHA2DS2-VASc score (6 points) than in those with the lowest score (1 point). Importantly, the occurrence of NOAF after MI was associated with an increased risk of IS. The IS subdistribution hazard was 2.1-fold higher in MI survivors who developed NOAF during the study follow-up. Despite the potential influence of cofounding factors, our results suggest PCI to be the more favorable method for revascularization in relation to NOAF occurrence, as CABG was associated with a higher NOAF incidence at one year (1.9-fold) and, interestingly, at ten years (1.3-fold) as well.Our results found that the CHAA higher NOAF rate after MI compared to the general population has been well documented, with incidences ranging from 6% to 21% at five years post-MI . Our fin2DS2-VASc score used to predict NOAF in patients with a history of either STEMI  or non-STEMI. Our results show that the CHA2DS2-VASc score can indeed be used to identify at-risk patients to yield more targeted follow-ups and rhythm monitoring to detect AF. In 2014, Lau et al. studied the usefulness of the CHA2DS2-VASc score in predicting NOAF and IS in post-STEMI patients [2DS2-VASc point when considering whether to initiate anticoagulation in those with paroxysmal AF. AF develops in 20–40% of patients within the first week after CABG. Revascularization by CABG has been found to be associated with higher NOAF rates compared to patients who undergo PCI [Several independent risk factors for AF have been identified in the general population, but fewer have been identified for MI survivors . Most stpatients . Conversergo PCI . Furtherergo PCI . Despiteergo PCI . ConsequPatients with a history of MI are also at an increased risk of IS during the first three months after MI . PreviouThe incidence of IS was lowest in patients who underwent revascularization by PCI compared to those treated with CABG or medical therapy. This has also been suggested previously . In addi2DS2-VASc components [2DS2-VASc score during follow-up. Atrial fibrillation was detected using registries of specialist health care and death certificates, [Our study has strengths and limitations. The major strength is the population-based design, which included nearly all patients with MI in Finland during a 14-year period. The major limitations are the retrospective design and use of registry data. We did not have access to more detailed clinical data and lacked blood pressure, laboratory, imaging, and angiographical findings. Although we used an extended version of a previously validated method to detect CHAmponents , it is pficates,  but we dficates, . The pro2DS2-VASc scores may be helpful in future efforts aimed at NOAF detection to prevent IS.Our study of 70,922 patients showed that the CHA2DS2-VASc-score is a feasible tool for long-term NOAF risk stratification in MI survivors. In addition, our results show that CABG is associated with increased long-term NOAF incidence after MI. The hazard of IS was roughly two-fold higher for patients with an NOAF diagnosis compared to those without. These results indicate that simple and clinically easily applicable risk stratification based on CHA


The effects of FEP content on the morphology, roughness, wettability, pore size, and mechanical property of PSF/FEP MMMs were characterized by scanning electron microscope, confocal microscopy, contact angle goniometer, mercury porosimetry, and tensile testing instrument, respectively. When the FEP content was 9 wt%, the average roughness of M-4 reached 0.712 μm. Meanwhile, the water contact angle (CA) and the water sliding angle (SA) was 153.3° and 6.1°, respectively. M-4 showed super hydrophobicity with a micro- and nanoscale structure surface. Then, M-4 was used for separating of water-in-oil emulsion, showing high separation efficiency for water-in-kerosene and water-in-diesel emulsions of 99.79% and 99.47%, respectively. The flux and separation efficiency changed slightly after 10 cycles. Therefore, this study indicated that the obtained PSF/FEP MMM with super hydrophobic surface could be used for efficient water-in-oil emulsion separation.Polysulfone (PSF)/fluorinated ethylene propylene (FEP) mixed matrix membranes (MMMs) with super hydrophobic surface were successfully fabricated  The PSF/FEP membrane with super hydrophobic and super oleophilic surface had an outstanding separation performance for water-in-oil emulsion. The separation of emulsified oil/water mixtures is always difficult and challenging because the droplets are usually stable and nanoscale.3 Conventional techniques such as skimming, ultrasonic separation, air flotation, gravity processing, coagulation–flocculation, and chemical de-emulsification method are often limited by their low separation efficiency, high energy cost, complex separation devices and secondary pollution etc.4–6 Very recently, membrane filtration has become an important technology in the oil/water separation field because of its many advantages, such as high filtration efficiency, great selectivity, low energy cost, and environmental friendly.7–9Environmental pollution has got increasingly serious with the rapid expansion of industrialization. Emulsified oil/water mixtures are generated in diverse industries, such as petrochemical, food, textile, leather, steel, aluminum, and metal finishing, the direct discharge of which will threaten the environment and human health.10 A series of “oil-removing” membranes have been fabricated for highly efficient oil/water separation by designing superhydrophobic and superoleophilic surfaces. Li et al.11 immobilized Ag nanocluster on nano-fibrous membrane for oil/water separation. Cao et al.12 modified stainless steel mesh using chemistry and Michael addition reaction by n-dodecyl mercaptan for efficient oil/water separation. Zhang et al.13 fabricated superhydrophobic and superoleophilic polyester materials by one-step growth of silicone nanofilaments onto the textile via chemical vapor deposition of trichloromethylsilane for oil/water separation. These materials usually show ultrahigh permeation flux for the separation of immiscible oil/water mixtures. Nevertheless, these materials could not effectively separate emulsified oil/water mixtures because their pore sizes (about tens of micrometers) are much larger than the droplet sizes of the emulsion .14Generally, the membranes are classified as the hydrophobic–oleophilic membrane and hydrophilic–oleophobic membrane for “oil-removing” and “water-removing” process, respectively.2 Meanwhile, the oil quickly penetrates through the membrane and water is retained above. Generally, the emulsion separation efficiency increases with the improvement of hydrophobicity.15Superhydrophobic surface is one of the most important parameters for the separation of water-in-oil emulsion. When emulsion droplets touch the superhydrophobic surface, a de-emulsification process could proceed immediately on the membrane surface due to the different wettability for oil and water.11,13,16–20 which can be challenging for the large-scale membrane fabrication.The preparation of the superhydrophobic membranes for the separation of water-in-oil emulsion usually requires expensive materials, strict conditions , and complex processing methods including plasma etching, chemical vapor deposition, electrodeposition, calcination and the use of templates,21–24 nanofiltration,25,26 and matrix membranes27–29 due to its outstanding properties of thermal stability, chemical resistance, high mechanical strength, and wide aperture adjustment range.30 Hydrophobic SiO2 nanoparticles have the features of small particle size, narrow particle size distribution, and large surface area,31 which play an important role in making a rough and superhydrophobic surface.15,32–34 FEP resin has lower surface free energy due to its chemical compositions, which is usually used to prepare and modify for hydrophobic membrane.35–37PSF is a classical membrane material for the preparation of ultrafiltration,2 nanoparticles and micro-nanometer scale FEP particles were introduced into PSF casting solution to fabricate micro- and nanoscale hierarchical structures on the membrane surface. Meanwhile, the surface energy of membrane decreased due to the existing of FEP on the surface, which was beneficial to fabricating of superhydrophobic surface, and there were obvious improvements on the comprehensive performance of the as-prepared MMMs. The effects of FEP content on MMMs' performances in terms of hydrophobicity, mechanical strength, permeability, and separation performance of water-in-oil emulsion were investigated respectively.Herein, we report the fabrication of PSF/FEP mixed matrix membranes (MMMs) for water-in-oil emulsion separation by using environmentally friendly and low cost chemicals as well as under simple and easy conditions to meet the need on an industrial scale. SiO2.2.1co-hexafluoropropylene) resin (6100) was purchased from DuPont Co., Ltd (U.S.A.). Hydrophobicity SiO2 particles (average particle size 40 nm) were purchased from Guangzhou GBS High-tech & Industry Co., Ltd. N,N-Dimethylacetimide  was obtained from Samsung Fine Chemical Co., Ltd. . Di-n-octylo-phthalate (DOP), ethyl alcohol, Span80  = 4.3), kerosene and diesel were bought from Tianjin Fengchuan Chemical Reagent Technologies Co. Ltd.Polysulfone (PSF) was purchased by Solvay Group-Solvay (Shanghai) Co., Ltd. Poly by measuring the contact angle of water and oil. The water droplet and oil droplet about 2 μl were dropped on the membrane surface, respectively. The porosity of PSF/FEP MMMs were measured by the gravimetric method. The porosity (ε) was used to calculate by the following W1 is the weight of the wet membrane, W2 is the weight of the dry membrane, D1 is the n-butyl alcohol density, and D2 is the density of PSF/FEP MMMs.PSF/FEP MMMs had large roughness, which was not suitable for quantifying by Atomic Force Microscope (AFM). So, surface roughness of the membrane was measured by Confocal Scanning Microscope . The samples were cut into 5 mm (width) × 5 mm (length) test strips and then were stick on the smooth glass. The morphologies of membrane surface and cross-section were observed using field emission scanning electron microscopy . The samples were coated in gold before testing. The mechanical properties of PSF/FEP MMMs were measured by YG-061-1500 tensile tester at room temperature. The samples were cut into 5 mm (width) × 50 mm (length) test strips. Each sample was measured 5 times at the tensile rate of 2 mm minJ is the oil flux (L m−2 h−1), V is the oil total volume (L), A is the membrane area (m2), t is the operating time (h).The pore size and pore size distribution of membranes were measured by mercury porosimetry . The oil flux of PSF/FEP MMMs were measured by a laboratory device . The sam2.4W is the separation efficiency (%), C0 is the water content of fresh water-in-oil emulsions (ppm), C1 is the water content of collected filtrate (ppm).Surfactant-stabilized water-in-oil emulsion is stable and difficult to separate. In this work, we selected surfactant-stabilized water-in-oil emulsion to test the separating performance of PSF/FEP MMMs. Surfactant-stabilized water-in-oil emulsions were prepared by adding 0.5 g Span80 into 100 g oil (kerosene and diesel), stirring 30 min, then adding 5 g water dropwise slowly, and vigorously stirring for more than 3 h. The water-in-oil emulsions of kerosene and diesel were stable for separation experiment. The separation experiment was conducted by the laboratory device as shown in 3.3.112 One is to create a rough structure on the hydrophobic surface,38 and the other is to modify a rough surface by low surface free energy materials.39 However, it has demonstrated that the –CF3– terminated surface possessed the lowest free energy, while the maximum water contact angle could only reach about 120° on flat surface.40 As described in Wenzel equation,41 it could be known that the surface roughness was also a key factor for super hydrophobic surface. The membrane surface roughness was showed in Conventionally, super hydrophobic surface has been produced mainly by two ways.3.22 nanoparticles and micro-nanometer scale FEP particles. When the FEP content was 9 wt%, many papillae were clearly found on the M-4 surface with average diameter about 145 nm. These micro- and nanoscale structures improved the membrane surface roughness and hydrophobicity. However, when the content of FEP was up to 12 wt%, FEP particles were inclined to aggregate, which destroyed the structures of membrane surface. Morphologies of the membrane cross-sections were shown in Morphologies of PSF/FEP MMMs with different FEP contents were observed by FESEM. As shown in The thickness of PSF/FEP MMMs was shown in 3.3The mechanical properties of PSF/FEP MMMs were discussed and shown in 3.4322. When FEP concentration was 9 wt%, the micro- and nanoscale hierarchical structures were fabricated on the M-4 surface. The CA of M-4 was about 153.3°. And the SA of M-4 was about 6.1° lower than 10° ) determined by placing a water droplet on its surface, which was inclined to tilt gradually until water droplet started to roll, as shown in Super hydrophobic and super oleophilic membrane surface is one of the most important properties for the efficient separation of water-in-oil emulsion. Generally, surfaces with a water contact angle (CA) larger than 150° and a water sliding angle (SA) lower than 10° are considered to be super hydrophobic surfaces.As shown in The oil contact angle of PSF/FEP MMM was tested using kerosene on the M-4 surface. As shown in Furthermore, the water-adhesion property of M-4 surface was also characterized. 3.5Porosity of the hydrophobic membrane was an important property for oil flux in membrane distillation. The porosities of PSF/FEP MMMs were measured using the gravimetric method, and the porosity results were shown in The average pore size of PSF/FEP MMMs was shown in 3.6−2 h−1 and 668.8 L m−2 h−1 for kerosene and diesel, respectively. The membrane had higher porosity and oleophilic property, when FEP concentration was 9 wt%. The flux of diesel was higher than kerosene because the viscosity of kerosene was lower. As shown in −2 h−1 and 435.8 L m−2 h−1, respectively.The pure oil fluxes of PSF/FEP MMMs for kerosene and diesel were tested and shown in 3.742–45 To study the separation performance further, the membrane was exposed to the water-in-oil emulsion for a long time, which would cause unavoidable membrane fouling, leading to the reduction of membrane's service life and the separation efficiency. During a separation cycle, the membrane was immersed 20 mL ethanol for 2 h and dried in air. The process was repeated ten times and the oil purity of filtrate was counted each time. As demonstrated in We made water-in-kerosene and water-in-diesel stabilized emulsions with surfactant to conduct water-in-oil emulsion separation experiment. The comprehensive properties of PSF/FEP MMMs were shown in 4.2 nanoparticles and micro-nanometer scale FEP particles fabricated unique micro- and nanoscale hierarchical structures on the membrane surface. With increasing FEP content, the structures of membrane surface changed obviously. When the content of FEP was 9 wt%, the average roughness of M-4 reached 0.712 μm. Meanwhile, the CA and the SA of M-4 was 153.3° and 6.1° respectively, showing super hydrophobicity. The fluxes of kerosene and diesel were stable about 246.7 L m−2 h−1 and 435.8 L m−2 h−1, respectively. And the separation efficiency of water-in-kerosene emulsion and water-in-diesel emulsion reached 99.79% and 99.47% after 10 cycles with an outstanding separation performance.PSF/FEP MMMs were prepared by NIPS method successfully. SiOThere are no conflicts of interest to declare.RA-008-C8RA00055G-s001


To compare the quantitative and qualitative optical outcomes of single-step transepithelial photorefractive keratectomy (TPRK) and off-flap epipolis-laser in situ keratomileusis (Epi-LASIK) in moderate to high myopia.In this prospective self-control study, we included patients with moderate to high myopia who were randomized to undergo TPRK in one eye and Epi-LASIK in the other eye. Twelve-month follow-up results for visual acuity, refraction, ocular high-order aberrations, contrast sensitivity, postoperative pain, epithelial healing, and haze grade were assessed.A total of 64 eyes (32 patients) were enrolled in the study. More eyes completed re-epithelialization in the TPRK group than in the Off-flap Epi-LASIK group 3–4 days postoperatively, while all eyes completed re-epithelialization by 7 days. More eyes achieved a visual acuity (both UDVA and CDVA) of better than 20/20 in the TPRK group than in the Off-flap Epi-LASIK group. The ±0.50 D predictability for correction of the spherical equivalent (SE) was higher in the eyes of the TPRK group (91%) than in those of the off-flap Epi-LASIK group (80%) 12 months after surgery. No significant differences in ocular aberrations, including coma, spherical, and trefoil, were found between the two groups at 12 months. There were also no significant differences in visual acuity, contrast sensitivity, pain, and haze grading between the two groups.Both TPRK and off-flap Epi-LASIK are safe, effective, and predictable treatments for moderate to high myopia with comparable surgical outcomes.ClinicalTrial.gov .This study was retrospectively registered on The online version contains supplementary material available at 10.1186/s12886-022-02443-6. Photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK) were introduced decades ago , 2, and A key aspect of modern surface ablation is epithelial removal. Laser-assisted sub-epithelial keratectomy (LASEK), a technique which allows for the separation of the epithelium using 20% alcohol, was introduced in 2001 . And, inAnother alternative technique for epithelial removal is transepithelial PRK (TPRK), in which epithelial removal is performed with laser phototherapeutic (PTK) ablation, followed by laser refractive ablation of the stroma. However, several studies have reported that two-step TPRK using different laser platforms produces variable results –11. ExciTherefore, in this study, we evaluated the long-term outcomes of single-step TPRK compared to off-flap epi-LASIK in moderate to high myopia, including qualitative and quantitative parameters, and performed vector analysis of astigmatism.Patients with myopia (up to − 9.75 D) with or without astigmatism (up to 3.5 D) were enrolled in a prospective interventional study. The study was approved by the Wenzhou Medical University Institutional Review Board and adhered to the tenets of the Helsinki Declaration. Patients received a full explanation of both procedures and provided written informed consent. Patients were randomized to have TPRK in one eye and Epi-LASIK in the other eye using a random number table generated by Microsoft Excel.Inclusion criteria included: 18 years of age or older; a corrected distance visual acuity (CDVA) in logarithm of minimal angle of resolution (LogMAR) of 0.10 or better; refractive error stabilized for at least 1 year; and discontinuation of contact lens use for at least 2 weeks. Exclusion criteria included: the presence of corneal scars, keratoconus, glaucoma, retinal diseases, or a history of corneal or intraocular surgery.All patients underwent comprehensive ophthalmological examinations, including uncorrected (UDVA) and corrected distance visual acuities (CDVA), manifest and cycloplegic refraction preoperatively, intraocular pressure, an anterior and posterior segment examination, corneal topography measured by a Scheimpflug scanning-slit topographer , and contrast sensitivity under photopic and mesopic conditions  with correction by spectacles and ocular wavefront aberrometry . These examinations were repeated 1, 3, 6, and 12 months after surgery.Both single-step TPRK and off-flap Epi-LASIK were performed with the Amaris 750 S  using integrated ORK-CAM software, with the right eye operated on first in all cases. Local anesthetic was applied for the ablation procedure, using proparacaine hydrochloride 0.5% drops  three times every 5 min.In the single-step TPRK procedure, transepithelial corneal epithelial removal was performed using the ORK-CAM software module mode followed by stromal excimer laser ablation in a single step based on an aberration-free and aspheric profile. The area of epithelial removal was defined as the area of the total ablation zone. The software compensated for the energy loss at the slope of the peripheral cornea. In the off-flap Epi-LASIK group, the epithelium was removed using an epikeratome  with a diameter of 9 mm, the stromal ablation was performed with Amaris 750 s excimer laser. After ablation, 0.02% mitomycin-C was applied to the stromal bed for 15–30 s and then rinsed with a balanced salt solution. A bandage contact lens  was placed on the cornea at the end of the surgery. All procedures were performed by the same surgeon (XCC).Patients were prescribed levofloxacin 0.5% eyedrops  and pranoprofen 0.1% eye drops  four times a day for the first 3 days after surgery. After removing the contact lens, tobramycin-dexamethasone eye drops  were administered four times a day for 2 weeks, followed by 0.1% fluorometholone eye drops  which were used four times a day for the next 2 weeks, after which they were tapered every week to once daily. Preservative-free artificial tears every 2 hours were prescribed until complete re-epithelialization, and then reduced to four times a day for 6 months.Epithelial healing was observed daily from the third postoperative day, and the contact lens was removed when corneal re-epithelialization was completed. Subjective pain scores were evaluated on the third day according to a predetermined scale ranging from 0 to 5 as follows: 0, no pain or discomfort; 1, photophobia and tears; 2, photophobia and tears with mild pain; 3, photophobia and tears with moderate pain that does not require oral medication; 4, photophobia and tears with severe pain that oral medication can relieve; 5, photophobia and tears with severe pain that oral medication cannot relieve. The corneal haze grade was recorded according to the methods reported by Fantes et al. : 0, no hp < 0.05.Statistical analysis was performed using SPSS software . The Kolmogorov-Smirnov test was used to check the normal distribution of the variables. Student’s t-test or Wilcoxon rank sum test was used based on the normality of the data. Statistical significance was set at P < .001, Table In total, 38 patients were initially enrolled in the study. Thirty-two patients finally completed the 12-month follow-up, and six patients were lost to follow-up. No significant difference was noted between the groups at baseline with respect to spherical equivalent (SE), CDVA, central corneal thickness (CCT), or epithelial thickness. The optical zones between the groups were not significantly different. However, the central ablation depth and total ablation zone were significantly higher in the TPRK group compared to the Off-flap Epi-LASIK group (both P = .372).More eyes completed re-epithelialization in the TPRK group 3 days  and 4 days  after surgery than in the off-flap Epi-LASIK group. Nevertheless, all eyes underwent re-epithelialization by 7 days. There was no significant difference in median pain score (range) between the two groups  of the eyes in the TPRK group and 63.33% (n = 19) eyes in the off-flap Epi-LASIK group gained one or two lines of improved CDVA . Furthermore, no significant differences in UDVA or CDVA were noted between the groups  better than 20/20 in the TPRK group than in the Off-flap Epi-LASIK group  of 0.996 and 0.9717, respectively, and 0.998 and 0.9594, respectively, in the off-flap Epi-LASIK group  was higher in the eyes of the TPRK group 91%) compared to those of the off-flap Epi-LASIK group (80%) 12 months after surgery  of, 0.87, and 0.68, respectively, in the TPRK group  in the off-flap Epi-LASIK group exhibited postoperative cylinders of 1.00 D or less  12 months after surgery. In the eyes of the off-flap Epi-LASIK group, there was a significant decrease in ocular spherical and trefoil aberrations (both P ≤ .012) 12 months postoperatively. The difference in ocular higher-order aberrations (HOAs) between the two groups was not significant at 12 months after surgery. By 12 months, the photopic and mesopic contrast sensitivity was not significantly different between the groups , nine eyes (28.1%), and four eyes (12.5%) 1, 3, and 6 months postoperatively, respectively. In the TPRK group, 0.5 or 1.0 haze was found in 17 eyes (53.1%), 10 eyes (31.3%), and six eyes (18.8%) 1, 3, and 6 months postoperatively, respectively. There were no significant differences between the groups.In this self-controlled study, we investigated the surgical outcomes of TPRK and off-flap Epi-LASIK in patients with moderate to high myopia. The visual outcomes indicated that both procedures were safe and effective for the correction of moderate to high myopia. There were no significant differences in postoperative visual acuity, efficacy, and safety indices as well as photopic and mesopic contrast sensitivity between the two groups. The ±0.50 D predictability for correction of the SE refraction was higher in the TPRK group than in the Off-flap Epi-LASIK group 12 months after surgery. Additionally, no significant differences in ocular HOAs  were noted between the groups.n = 18) of the eyes in the TPRK group and 63.33% (n = 19) eyes in the off-flap Epi-LASIK group gained one or two lines of improved CDVA. The ±0.50 D predictability for correction of the spherical equivalent (SE) was higher in the eyes of the TPRK group (91%) compared to those of the off-flap Epi-LASIK group (80%) 12 months after surgery. We showed better performance mainly because the included eyes almost with regular astigmatism less than 3.50 diopters, and amblyopia was excluded. Furthermore, TPRK has been modified several times since its introduction in 2009. Another study by Arba et al. [Several previous studies have indicated that TPRK in myopic eyes is safe, effective and predictable , 17. Xi a et al.  reportedRegarding corneal haze, no significant difference was noted at any postoperative interval. By 6 months postoperatively, no eyes in either group had a haze over the score of 0.5. Haze can be seen after surface ablation in most cases as a mild anterior stromal opacity. It is considered to be the product of the normal healing process, which peaks at 3 months and fades at 6 months . Haze foIn our study, TPRK eyes showed faster re-epithelialization compared to Epi-LASIK eyes, but no significant difference was noted which is similar to findings from previous studies. For example, Aslanides et al.  found thIn reflecting the quality of vision in daily life situations, more attention should be paid to contrast sensitivity and wavefront aberrations rather than visual acuity alone. Ocular high-order aberrations significantly decreased 12 months after surgery in both TPRK and off-flap Epi-LASIK groups, but the difference between the two groups was not significant. Corneal refractive surgery could correct low-order aberrations such as myopia and astigmatism, and improve UDVA, but several studies have reported the increasing amount of HOAs postoperatively and a high correlation between HOAs and visual performance , 28, 29.The current study had some limitations. First, the sample size was relatively small. A large population size could increase the power to detect significant differences. Second, the follow-up duration was relatively short. Therefore, future studies incorporating a longer follow-up of visual performance between TPRK and off-flap Epi-LASIK are recommended.We conclude that both TPRK and off-flap Epi-LASIK are safe, effective, and predictable for treating moderate to high myopia. These two types of surgical techniques have comparable epithelium healing time, pain scores, corneal haze grade, and visual outcomes.Additional file 1.


Pseudomonas sp. L01, using response surface methodology (RSM). Optimal conditions yielded a crude biosurfactant yield of 8.43 g/L in a flask. Our work provides a high-throughput approach to the isolation and screening of biosurfactant-producing bacteria, as well as other functional bacteria in a wide range of fields.Biosurfactants are a group of surface-active compounds that can be produced by diverse microorganisms. They have been widely used in various industrial fields. Reducing production costs, improving efficiency, and collecting more diverse producing strains have become major challenges in the biosurfactant industry. These challenges could be overcome by screening for more diverse and efficient biosurfactant-producing strains. The conventional methods for the isolation and functional characterization of microorganisms are laborious and biased toward fast-growing or strongly competitive microorganisms. Here, we established a high-throughput approach of single-cell-based cultivation and functional characterization of biosurfactant-producing bacteria (SCCBB). This approach combines single-cell cultivation with the detection of optical distortions. Using this approach, we isolated 431 strains with biosurfactant production potential from petroleum-contaminated soil and oilfield-produced water. The surfactant production capabilities of the strains were subsequently validated using surface tension measurements, TLC, and CMC measurements. To investigate the industrial production potential, we optimized the production conditions of a representative glycolipids-producing strain,  Biosurfactants are a group of amphiphilic surface-active compounds mainly including glycolipids, lipopeptides, lipoproteins, lipopolysaccharides, and phospholipids, which can be produced by diverse microorganisms, including bacteria, yeast, and fungi ,2,3,4,5.Pseudomonas aeruginosa and Bacillus subtilis are the most attractive for commercial production due to their superior ability to produce rhamnolipids and surfactin, respectively. Additionally, many hydrocarbon degraders produce biosurfactants during hydrocarbon metabolisms, such as Rhodococcus, Dietzia, Acinetobacter, and Marinobiacter, which are thought to have potential in bioremediation and microbial enhanced oil recovery (MEOR) [Pseudomonas aeruginosa, is also a potentially pathogenic strain that could pose a challenge to biosecurity. Moreover, limited structural variation hinders the application of biosurfactants in more fields. These challenges could be overcome by screening new compounds from diverse biosurfactant producers.Benefiting from efforts to isolate and screen biosurfactant-producing microorganisms, a large number of producer strains have been identified. Of these microbial strains, members of the species y (MEOR) ,14,15. WConventional methods for isolating and culturing microorganisms are laborious, time-consuming, and biased toward those fast-growing or strongly competitive microorganisms ,17. The Developing fast and high-throughput methods for the detection of surfactant activity is also a challenge for the efficient collection of biosurfactant-producing bacteria. The conventional methods include surface and/or interfacial tension measurement, axisymmetric drop shape analysis profile (ADSA-P), glass-slide test, and the oil spreading method . HoweverHere, we established a high-throughput culturomics approach of single-cell-based cultivation and functional characterization of biosurfactant-producing bacteria (SCCBB). This approach combines high-throughput single-cell culture with the detection of optical distortions caused by suspensions containing biosurfactants. We validated this approach by successfully isolating biosurfactant-producing bacterial strains from soil and oilfield-produced water. The biosurfactant production conditions of a representative strain were optimized using the response surface methodology (RSM) method to test potentials in industrial applications.Two petroleum-contaminated soil samples  and an oilfield-produced water sample were collected in this work. Oil-soil-1 was an artificial petroleum-contaminated soil as described previously . Oil-soiThe oilfield-produced water was collected from the Ba51 fault block in the Baolige oilfield  located in the central part of Inner Mongolia, China, in March 2019. The samples were transported to the laboratory in ice within 48 h. The produced water was then filtered twice with gauze and collected for further experiments.4∙7H2O, 0.13 g/L K2HPO4, 0.22 g/L CaCl2∙2H2O, 0.17 g/L K2SO4; pH 7.0), TSB medium , and mineral medium with oilfield-produced water 2SO4, 5.0 g/L KCl, 0.2 g/L MgSO4∙7H2O, 0,05 g/L CaCl2∙2H2O, 10 mL trace element solution SL-4 , dissolved in 1 L of oilfield-produced water; pH 7.0).Four kinds of media were used for isolation, i.e., the LB medium , TYG medium .The total number of culturable cells in the sample was first calculated using the traditional plate counting method. The optimal dilution level can then be determined by a Poisson distribution, where most wells are expected to receive either one or two cells. For example, the cell suspension should be serially diluted to the theoretical cell density of 24 CFU/mL for cultivation in a 384-well plate (60 μL of sample in each well). At this dilution level, approximately 24.3% of the wells in a 384-well plate are expected to be blank. The actual dilution level should then be modified based on the initial culture in a 384-well plate, where 24% to 30% of the cells were clear after 7 days of cultivation.600 of the culture in the microplate was detected using a microplate reader to identify wells with successful culture. Then, 10 μL of supernatant of each well with turbid cultures was added to 90 μL of pure water in a 96-microwell plate. Distilled water was used as a negative control. The plate was then viewed with a sheet of grid-line paper underneath. Wells that showed optical distortions of the grid were thought to have surfactant activity and were selected for further analysis.The surfactant activity of each isolate was determined using a microplate assay . The OD6https://www.ezbiocloud.net/, accessed on 25 January 2021). A phylogenetic tree was constructed using the neighbor-joining method in MEGA X software [DNA was extracted from each colony on LB agar plates using the DNA isolation kit . The bacterial 16S rRNA gene was amplified by polymerase chain reaction (PCR) using universal primers 8F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The PCR process was performed using 2 × Taq Master Mix  with a three-step PCR program . The bacterial 16S rDNA sequences obtained were then aligned with known 16S rDNA sequences in EzBioCloud  based on surface contact angle [The strain was first grown for 24 h at 30 °C in LB liquid medium by shaking at 220 rpm. The cells were then washed twice with PBS to make an inoculum. The inoculum was then transferred into the fresh LB medium containing 1/10 peptone with initial ODct angle . Each sa2O . The resulting spots on the TLC were visualized using the spray reagent ninhydrin-acetone  for lipopeptide and anthrone-concentrated sulfuric acid  for glycolipid [The cells were cultured in an LB medium containing 1/10 of the peptone described above. After culture, each fermented broth was centrifuged at 5000 rpm for 10 min. The resulting supernatant was acidified to pH 2 with 6 M HCl and placed at 4 °C overnight. The crude biosurfactant extract was obtained after centrifugation at 8000 rpm for 15 min at 4 °C to remove the supernatant and dry at 90 °C to constant weight. The detection of the biosurfactant extraction was performed by thin-layer chromatography (TLC). The solvent system used was chloroform:methanol:Hycolipid .3, and (NH4)2SO4) on the yield of glycolipids were detected. The minimal medium (pH 7.0) consisted of 10 g/L Na2HPO4∙12H2O, 10 g/L KH2PO4, and 0.5 g/L MgSO4, which was supplemented with different carbon sources and nitrogen sources to evaluate the effects of carbon and nitrogen sources. After the carbon source and nitrogen source were determined, the medium was optimized using response surface methodology (RSM). The RSM experiment was designed using the Design Expert software . All experiments were performed in an Erlenmeyer flask with 50 mL of media.For biosurfactant production optimization, the effects of different carbon sources  and nitrogen sources . The data were statistically analyzed using a two-tailed unpaired Student’s t-test, where a Conventional methods for isolating and identifying biosurfactant-producing bacteria are inefficient and time-consuming, often relying on the isolation of colonies in a petri dish and functional characterization of scaled-up cultures in a flask. Moreover, cultivation based on colony selection suffers from severe cultivation bias due to the different growth fitness of various species. To overcome this problem, we established a high-throughput culturomics approach of single-cell-based cultivation and functional characterization of biosurfactant-producing bacteria (SCCBB). The SCCBB approach combined single-cell cultivation and high-throughput screening based on the detection of the optical distortion through the supernatant containing surfactants . In thisTraditional methods to detect surfactants, such as surface tension measurements and oil dispersal assays, are time-consuming and limit high-throughput screening of biosurfactant-producing bacteria. Here, we screened the surfactant activity from the cultures using a microplate method . The culLysinibacillus. Moreover, using the agar plate method, we failed to isolate strains belonging to the genus Pseudomonas, which are well-known biosurfactant-producing bacteria. It has been reported that the genus Lysinibacillus is fast-growing, stress-tolerant, and can induce the abnormal biofilms of other species by signaling molecules [Lysinibacillus, tend to be over-represented in culture conditions, suggesting that differences in growth fitness can lead to a cultivation bias when using the agar plate method.To assess the cultivation bias of the SCCBB method, we isolated twenty-one potential biosurfactant-producing bacterial strains from a petroleum-contaminated soil sample using this method. These strains belonged to eight genera . As a coolecules ,31,32. CPseudomonas , Lysobacter (21.6%), Bacillus (7.9%), Microbacterium (5.8%), Sphingopyxis (5.3%), and Brucella (5.1%). Of these genera, Pseudomonas strains isolated from all samples, accounted for nearly 70% of the abundance in oilfield-produced water. This result is consistent with the microbial compositions of oil reservoir microbiomes, which showed that Pseudomonas was the most abundant genus in the oilfield-produced water samples [Acinetobacter and Pannonibacter have also been isolated from the oilfield-produced water sample. Lysobacter, in contrast, has only been isolated from soil samples with relatively high abundances.Three samples, including two petroleum-contaminated soil samples and one oil-produced water sample, were selected for the isolation of biosurfactant-producing bacteria using different media using the SCCBB method. After 7 days of single-cell cultivation, all wells were screened by optical distortion. A total of 431 isolates with biosurfactant-producing potentials were obtained A. Based  samples . In addiBacillus. In addition, the strain L01 could reduce the surface tension of the medium to 43.16 ± 0.14 mN/m, representing the most active strain in the genus of Pseudomonas.Based on the 16S rRNA gene sequences, all 431 isolates can be assigned to 99 phylotypes . We randomly selected one candidate strain for each phylotype for further analysis. To assess the capacity of the isolates to produce biosurfactants, we measured the surface tension of the culture supernatant for each candidate strain. The results showed that 42 strains significantly reduced the surface tension of the culture medium A, which Pseudomonas sp. L01 and Bacillus sp. N110). Note that the cultivation conditions and growth rate for the two selected strains are different, so the periods for measuring growth and biosurfactant production are different for the two strains. We cultured Pseudomonas sp. L01 in a minimal medium with 2% (w/v) glucose as the sole carbon source and monitored the acid-precipitated crude biosurfactants at different time points. The results showed that the growth of L01 entered the stational phase after 24-h culture when the crude biosurfactant started to accumulate quickly  and lipopeptides , respectively [To verify whether the decrease in surface tension was caused by the biosurfactants, we isolated and identified the surface active products from two representative strains with high surface activities . We first evaluated the effects of different carbon sources  and nitrogen sources 2SO4) on the yield of glycolipids. The yields of crude biosurfactants were determined in cultures with different concentrations of each carbon or nitrogen source at different time points. The results showed that yields of the crude biosurfactants in the carbon source test (2.5 g/L (NH4)2SO4) were used as the nitrogen source) reached the highest level when 30 g/L of glucose or more was used. The maximum yield in the nitrogen source test (30 g/L glucose was used as the carbon source) was 6.55 ± 0.53 g/L when 3.0 g/L NaNO3 was used.To further investigate the potential of the strains for the industrial production of biosurfactants, we optimized the biosurfactants yield of 43− concentration, pH, and culture time as variables. The two-level values of these variables and the analysis of variance are shown in 3 and the time of culture were significant variables in the production of biosurfactants, with positive effects on the yield of biosurfactants.The Plackett–Burman (PB) design was used to evaluate the most significant variables in the production of biosurfactants for strain L01. In addition to the carbon and nitrogen sources, we include the PO3, the cultivation time, and the concentration of PO43− as the factors for the further steepest ascent experiment  before RSM optimization.The combined effects of NaNOur plots . AccordiIn this work, we developed a high-throughput and function-oriented culturomics method for isolating and screening the biosurfactant-producing bacteria, which combined single-cell culture and optical distortion detection. Using this method, we isolated 431 strains with biosurfactant production potential from petroleum-contaminated soil and oilfield produced water. Since the microplate is well-compatible with automated instruments such as liquid handling systems, the throughput will be even higher. It can also be embedded in an integrated automated laboratory. Thus, our work provides an automatable solution for high-throughput isolation and screening of biosurfactant-producing bacteria, which also shed light on efficiently screening functional bacteria in other fields.


Life-space and frailty are closely linked to health-related quality of life and understanding their inter-relationship could indicate potential intervention targets for improving quality of life. We set out to examine the relationship between frailty and life-space and their relative impact on quality of life measures.Using cross-sectional data from a population-representative cohort of people aged ≥ 70 years, we assessed quality of life with the EuroQol Health Index tool (5-levels) (EQ-5D-5L). We also undertook a life-space assessment and derived a frailty index. Linear regression models estimated EQ-5D-5L scores (dependent variable) using life-space assessment, frailty index and interactions between them. All models were adjusted by age, sex, lifestyle, and social care factors.p < 0.01) and decreasing frailty . There was evidence of an interaction between life-space and frailty, where the steepest gradient for life-space and EQ-5D was in those with the highest frailty .A higher EQ-5D Index was associated with higher life-space (0.02 per life-space assessment score, 95%CI: 0.01 to 0.03, Individuals with the highest frailty were twice as likely to have higher quality of life in association with a larger life-space. Interventions designed to improve quality of life in frail older people could focus on increasing a person’s life-space.The online version contains supplementary material available at 10.1186/s12877-022-03355-2. Maintaining health-related quality of life (HRQoL) into older age is a key ambition for multidisciplinary healthcare teams , 2. QualLife-space is a rich and informative assessment of mobility with good construct validity, yet it is not yet well-established as a clinical tool . Higher We set out to examine the relationship between life-space, frailty, and their relative impact on overall quality of life in a population-representative cohort. We investigated variables related to quality of life and life-space, hypothesising that increasing life-space would be associated with improving quality of life to varying degrees between frail and robust older adults.We used data collected between 2018 and 2019 from the Delirium and Population Health Informatics Cohort (DELPHIC). Details have already been published –16. In bQuality of life was defined by the EuroQol Health Index tool (5-levels) (EQ-5D-5L) , which iThe life-space assessment is a self-reported measure of an individual’s independent mobility . It relaWe included health, social and lifestyle factors, such as frailty, frequency of contact with next of kin, and receiving a care package. We selected these based on previously reported associations with quality of life , 20 and We used a series of linear regression models to estimate the associations between our continuous outcomes  and covariates. We used median imputation for any data missing within the life-space assessment dimensions and multiple imputation (20 imputations) for other missing covariate data. We assessed interactions between life-space and frailty index scores by creating a parameter multiplying the two continuous measures, testing the model with and without this interaction term. To improve comparability and ease of interpretation, we transformed these into standardised z-scores (score-mean)/standard deviation). We used Stata version 16.1 for all analyses .n = 1152). Individuals in the middle tertile of EQ-5D Index (between 0.7 and 0.8) had a Visual Analogue Scale score of 79/100. Participants reported an average life-space of 66/120, broadly equivalent to someone who is able to leave their neighbourhood several times a week with the assistance of equipment. The mean frailty index was 0.15 (Table The mean age of the full sample was 78 SD 6.2), and 41% were men, and most individuals were educated to degree, or postgraduate level and had high-skill occupations , higher life-space  and decreasing frailty  (Table p < 0.01). Similar patterns were evident for the Visual Analogue Scale scores. Neither self-reported isolation, frequency of contact, nor distance from next of kin were associated with quality of life.A higher EQ-5D Index was associated with older age  and frailty  (Supplementary Table p < 0.01). There was a graded association between educational attainment and life-space, such that those with no qualifications had the lowest life-space .Lower life-space was associated with older age . This coefficient was the same as the association between life-space and EQ-5D, which translates to a doubling of the effect size for each SD of increasing frailty. The highest effect was in those with a high level of frailty compared to those with a low and medium level of frailty. Improving indoor and outdoor mobility (and frequency of being outdoors) in the middle range of life-space (-1SD to + 1SD), would be associated with a 0.1 point (one tertile) improvement in quality of life (Table p < 0.01); medium frailty , compared to low frailty .Additional file 3: Supplementary Table 3. Variables associated with HRQoL (with frailty in levels)Additional file 4: Supplementary Figure 1. Variables associated with life-space.


Voxel-based specific region analysis systems for Alzheimer’s disease (VSRAD) are clinically used to measure the atrophied hippocampus captured by magnetic resonance imaging (MRI). However, motion artifacts during acquisition of images may distort the results of the analysis. This study aims to evaluate the usefulness of the Pix2Pix network in motion correction for the input image of VSRAD analysis. Seventy-three patients examined with MRI were distinguished into the training group (n = 51) and the test group (n = 22). To create artifact images, the k-space images were manipulated. Supervised deep learning was employed to obtain a Pix2Pix that generates motion-corrected images, with artifact images as the input data and original images as the reference data. The results of the VSRAD analysis (severity of voxel of interest (VOI) atrophy, the extent of gray matter (GM) atrophy, and extent of VOI atrophy) were recorded for artifact images and motion-corrected images, and were then compared with the original images. For comparison, the image quality of Pix2Pix generated motion-corrected image was also compared with that of U-Net. The Bland-Altman analysis showed that the mean of the limits of agreement was smaller for the motion-corrected images compared to the artifact images, suggesting successful motion correction by the Pix2Pix. The Spearman’s rank correlation coefficients between original and motion-corrected images were almost perfect for all results . Pix2Pix generated motion-corrected images that showed generally improved quantitative and qualitative image qualities compared with the U-net generated motion-corrected images. Our findings suggest that motion correction using Pix2Pix is a useful method for VSRAD analysis. Alzheimer’s disease (AD) is the most common form of dementia , and hipThe following three methods have been proposed to correct motion artifacts in MRI images. The first method is to set up a motion correction, such as periodically rotated overlapping parallel lines with enhanced reconstruction, in the MRI imaging sequence . The secTo the best of our knowledge, there have been few studies using GAN and CNN motion-correction techniques for VBM . In partThe present study retrospectively analyzed 78 consecutive Japanese patients who visited the research hospital, Institute of Medical Science, the University of Tokyo from April 2013 to April 2020 and underwent MRI scans for suspected dementia. The Research Ethics Committee of the Institute of Medical Science, University of Tokyo, approved the study protocols (2019-41-1031), and the requirement for written informed consent was waived.Whole brain images were acquired using a 3.0 Tesla clinical MRI scanner  with Syngo MR E11 software and a 20-channel head matrix coil. The VSRAD images were acquired using a T1-weighted, magnetization-prepared rapid gradient-echo sequence with 176 sagittal slices . Following review by two experienced radiologists, five patients were excluded due to motion artifacts in their images, and the remaining 73 patients were divided into training  and test  groups. Ten percent of the training group was assigned to the validation group. The workflow of this study is illustrated in T1-MPRAGE ; VSRAD .et al. [In the present study, we used a computer with 3.6GHz/s (corei7–7700), 48GB/s memory bandwidth, and a GeForce GTX 1070 graphics card  with 8GB memory per board. Images with simulated motion artifacts were modeled in reference to the study of Lucilio et al. . The creUsing the same method with the training group, artifact images were generated for the test group with four fixed patterns 0_none , 1°_30, 2°_20, and 3°_10 . In addition, we created two patterns of intervals 1.5°_25, 2.5°_15  that were not trained with Pix2Pix.We used the Pix2Pix network to generate motion artifact corrected images. We used the Python 3.6.10 with Anaconda 3.0 distribution software , TensorFlow-GPU 2.1.0, and Keras 2.3.1 .For the Pix2Pix generator network, we used a U-Net-based network  with a sThe Pix2Pix discriminator network introduces PatchGAN that evaluates whether the original image and the motion-corrected image are equivalent in a small region. This network does not directly compare the two images. Instead, it splits each image into small regions of a specific size (patch), evaluates each pair , and finally performs binary classification. The use of PatchGAN has been reported to show high learning accuracy for high-frequency components of images . In the The learning parameters were trained using an Adam optimizer with a constant learning rate of 1e-4, beta_1 = 0.9, and beta_2 = 0.999 . The minIn the present study, the comparison between Pix2Pix and CNN was performed with U-Net, which was used in the generator network of Pix2Pix. The training parameters were set the same as for the Pix2Pix generation network, except that the mini-batch size was 16. The loss function for this network was set to Mean Squared Error.For quantitative evaluation, we used the structured similarity indexing method (SSIM) to compare the structural and feature similarities between the original images and the artifact or motion-corrected images. Analysis of SSIM was performed using MATLAB software R2021a.For qualitative evaluation, two diagnostic radiologists evaluated the artifact images and motion-corrected images (U-Net and Pix2Pix) generated in this study using the following 5-point Likert scale for the degree of motion artifact.5: No influence of movement4: Slightly affected by motion but does not affect analysis3: Lower limit of acceptable motion effect2: Motion influence is present and may affect the analysis.1: Cannot be analyzed due to the effect of motion.The radiologists were blinded to the image acquisition method and independently assessed the subjective image quality. The data were analyzed randomly to reduce recall bias.Multiplanar reconstruction was performed on the original image, artifact image, and the motion-corrected image in the 1-mm coronal plane. These three types of images were analyzed using the VSRAD advance software package . Three factors were analyzed:First, the severity of VOI atrophy was the Z-score of the severity of gray matter (GM) atrophy in the region of interest in AD, and it was calculated using the following equation:(severity of VOI atrophy) = /normal controls standard deviation (SD)).Second, the extent of GM atrophy referred to the extent of GM atrophy in the whole brain (extent of GM atrophy) = a percentage of voxels with a Z-score>2 compared with the whole brain.Third, the extent of VOI atrophy reflected the extent of GM atrophy in the VOI of AD, and it was calculated as follows:(extent of VOI atrophy) = ×100%) , 22.U test. The results of SSIM analysis were compared using paired t-tests, and qualitative image quality was compared using Wilcoxon signed-rank test. A weighted kappa test was used to examine the inter-observer agreements of qualitative image quality evaluation. A kappa value of 0.20 was defined as a slight agreement, 0.21–0.40 as fair agreement, 0.41–0.60 as moderate agreement, 0.61–0.80 as strong agreement, and 0.81–1.00 as almost perfect agreement. In the Bland-Altman analysis, the mean bias of the VSRAD analysis in the artifact vs. the original image, U-Net motion-corrected vs. the original image, and the Pix2Pix motion-corrected vs. the original image were plotted against the difference of the values, and the limit of agreement between the two measurements was defined as the mean±1.96 times SD of the difference with a 95% confidence interval. The results of VSRAD analysis were tested for severity of VOI atrophy, extent of GM atrophy, and extent of VOI atrophy. Four trained patterns  and two non-trained patterns  were tested. In addition, Spearman’s rank correlation coefficients were calculated for the VSRAD measurements of the artifact vs. the original images, U-Net motion-corrected images vs. original images, and the Pix2Pix motion-corrected vs. original images.The male-to-female ratio was compared between the training and the test groups using the Fisher test. Age, severity of VOI atrophy, extent of GM atrophy, and extent of VOI atrophy were compared between the training and the test groups using the Mann-Whitney These statistical analyses were performed using the EZR software . The sigThe number and mean±SD age of the patients in the training group was 51  and 75.24±12.16 years old, respectively. There were 22 patients in the test group , and the mean age was 75.00±11.88 years old. The mean values of severity of VOI atrophy, extent of GM atrophy, and extent of VOI atrophy were 1.23±0.87, 5.62±3.21, and 14.83±21.08 in the training group and 1.48±1.17, 5.77±2.99, and 25.96±32.56 in the test group. There were no significant differences in sex, age, severity of VOI atrophy, extent of GM atrophy, and extent of VOI atrophy between the training and the test groups .An example of the original image, artifact image, and motion-corrected image is shown in t-test with the same rotation angle and k-space interval to evaluate the SSIMs of the motion-corrected images, and we found that Pix2Pix was significantly higher than U-Net (p < 0.05). Qualitative evaluation revealed that motion-corrected images generated by U-Net and Pix2Pix from artifact-free images (0_none) were all point as 5. Furthermore, there was no difference in the qualitative evaluation between Pix2Pix and U-Net when the rotation angle was 1°_30 . For the 2°_20 and 3°_10 motion patterns, Pix2Pix showed a significantly higher score by both observers compared to U-Net . The other 1.5°_25 and 2.5°_15 motion patterns, showed a significantly different higher score by one observer . The weighted kappa test showed strong agreement between the two radiologists (k = 0.62).The results of the SSIM analysis for artifact images, U-Net motion-corrected images, and the Pix2Pix motion-corrected images against the original image are shown in vs. original images, and the Pix2Pix motion-corrected image against the original image are shown in Tables The results of the Bland-Altman analysis for VSRAD of the artifact image, U-Net motion-corrected images p < 0.05), extent of GM atrophy: 0.036–0.35 (p = 0.072–0.84 >0.05), extent of VOI atrophy: 0.64–0.76 (p < 0.05), all Spearman’s rank correlation coefficient). In contrast, the results of the U-Net and Pix2Pix motion-corrected image showed an almost perfect match with the original image with respect to the trained artifacts , Pix2Pix: 0.94–0.99 (p < 0.05), extent of GM atrophy, U-Net:0.91–1.00 (p < 0.05), Pix2Pix: 0.92–0.98 (p < 0.05), extent of VOI atrophy, U-Net:0.95–0.99 (p < 0.05), Pix2Pix: 0.94–1.00 (p < 0.05)) (p < 0.05), Pix2Pix: 0.87–0.97 (p < 0.05), extent of GM atrophy, U-Net:0.85–0.95 (p < 0.05), Pix2Pix: 0.88–0.97 (p < 0.05), extent of VOI atrophy, U-Net:0.91–0.96 (p < 0.05), Pix2Pix: 0.90–0.97 (p < 0.05)) ) , Fig 9, < 0.05)) .In the present study, the Pix2Pix network was trained by simulated artifact images as the input data and original images as the reference data to create motion-suppressed images (motion-corrected images) from artifact images. The results of the VSRAD analysis of the original image, the artifact image, and the motion-corrected image, were then compared. Bland-Altman analysis and Spearman’s rank correlation coefficient analysis revealed that the results of the VSRAD analysis of the motion-corrected image were more strongly correlated with the original image compared to the artifact image, suggesting that successful motion correction was obtained by Pix2Pix. Also, compared with U-net-based motion-corrected images, Pix2Pix-based motion-corrected images showed a generally higher image quality both subjectively and objectively.The artifact images used in this study simulated two patterns of motion: rotational motion in the vertical direction and the loss of signal due to anterior-posterior motion in the sagittal plane by filling the k-space with zeros. Several studies have simulated motion artifacts using a similar method to our study , 24. WitIn the present study, we compared motion correction between GAN and CNN using U-Net, which was used to generate Pix2Pix. U-Net learns the differences between input and output images as a loss function. Therefore, U-Net learns the relationship between two images by referring to the whole image. In contrast, Pix2Pix uses PatchGAN as a discriminator, learning the true or false pair relationship of the input image by referring to the patch image, and learning a generator whose discrimination accuracy is a loss function. For these reasons, the learning methods are different even though the same network structure is used to generate the images.Mode collapse is one of the reasons that GANs are generally considered to have poor learning stability . Only onLarge positive fixation biases were observed in the artifact images in the present study. A study of automated gray matter volume measurement reported that more significant motion artifacts resulted in smaller gray matter volumes . This suvs. artifact images showed a lower correlation for the extent of GM atrophy compared to the severity of VOI atrophy and the extent of VOI atrophy. The severity and extent of VOI atrophy in the VSRAD analysis set the medial temporal regions  as the regions of interest, while the extent of GM atrophy measured the gray matter of the whole brain. In the present study, the k-space was rotated from the center coordinate to create an artifact image. The severity and extent of VOI atrophy measures the medial temporal region close to the central coordinate, i.e., the region with the small movement. In contrast, the extent of GM atrophy measures the gray matter of the whole brain, including the gray matter with larger movement, resulting in a lower correlation with the original image. By contrast, the Spearman’s rank correlation coefficient between the original and the motion-corrected images by Pix2Pix were all very high: 0.87–0.99 for the severity of VOI atrophy, 0.88–0.98 for the extent of GM atrophy, and 0.90–1.00 for the extent of VOI atrophy, regardless of the rotation angle and k-space interval. This suggests that the present Pix2Pix network could learn to reduce artifacts stemming from both large and small movements.The results of the Spearman’s rank correlation coefficient analysis between the original We believe that the Pix2Pix network offers significant advantages in correcting motion artifacts in the VSRAD analysis. Firstly, the mean±SD of the severity of VOI atrophy in the healthy elderly group was reported to be 0.94±0.32 . In contOur study has several limitations. First, we used supervised learning in CNN and GAN training, a set of motion artifact images, and motion-less images. However, it was very difficult to acquire two types of images—one with motion and the other motion-less—from all AD patients. Therefore, the created artifact images were not real motion artifact images, so it remains unclear whether our findings results could be translated to clinical practice. Secondly, this study simulated vertical rotational and anterior-posterior-motion movements in the sagittal plane but did not examine left-right motion in the coronal plane. Thirdly, we did not evaluate the relationship between VSRAD score and severity classification of AD. Fourth, k-space under-sampling can reduce motion artifacts by minimizing the acquisition time. However, we did not acquire such images in this retrospective study, thus comparison between the image quality of under-sampled images and Pix2Pix motion-corrected images was not possible. Finally, the present study was performed in a single institution and the MR image data were obtained by a single machine. In the future, we would like to investigate the usefulness of similar motion correction techniques for data stored at other facilities and on other image databases.In conclusion, the VSRAD analysis tended to overestimate atrophy in images with artifacts. After reconstructing the artifact images using the Pix2Pix network to reduce motion artifact, the overestimation of atrophy was improved dramatically. We suggest that motion correction using Pix2Pix can be useful for VSRAD analysis.